This invention relates to a process for cloning luminescence genes. More specifically, this invention relates to a process for cloning luciferase structural genes of a bioluminescent microorganism, such as Vibrio harveyi, to produce a transformed microorganism capable of expressing bioluminescence.
It is generally recognized that information which determines the structure of proteins within a cell is contained within the DNA of that organism. Similarly, the information required for control of the synthesis of protein is resident within the DNA. As the genetic code is universal, the nucleotide base sequence within the DNA of one organism will code for production of the same amino acid sequence in another. Therefore, the transfer of DNA from one organism to another is the primary process involved in recombinant DNA technology. The procedure by which this process is accomplished in a laboratory, which is referred to as cloning, involves the formal insertion of fragments of DNA from one organism into a cloning vehicle and transfer of the cloning vehicle into a host cell. In this context, the cloning vehicle may be a plasmid DNA, which is a small circular piece of DNA, or any other DNA sequence which is able to replicate in a host cell.
The fragments of DNA used in cloning are generally derived from cleavage of the DNA molecule, which can be accomplished either by random shearing or by digestion with restriction endonuclease enzymes. Insertion of the DNA fragment in the cloning vehicle is generally accomplished by a process referred to as ligation. Where the cloning vehicle is a plasmid, the DNA ring is opened by digestion with a restriction endonuclease. The newly generated ends of the plasmid DNA are then joined with the ends of the DNA fragments to be cloned by the use of an enzyme such as T4 DNA ligase. I. R. Lehman, Science 186, 790 (1974).
One of the primary purposes of cloning DNA fragments is to obtain the expression of a given gene, such as the production of a protein encoded by that gene, in a different organism. Control of the production of that protein, however, requires the existence of a DNA structure referred to as a promoter sequence. The promoter sequence must be positioned properly relative to the inserted fragment of DNA to allow the binding of RNA polymerase from the host organism to the DNA such that messenger RNA molecules and corresponding protein molecules can be produced. The expression of a cloned gene in a host cell then requires, in addition to the cloned gene itself, a promoter sequence that will allow expression of that gene in the host organism. A problem of considerable importance in genetic engineering technology is the difficulty of obtaining a promoter sequence that promotes the expression of the desired genetic information.
Bacterial luciferase is an enzyme which catalyzes the light emitting reaction of naturally bioluminescent bacteria, such as Vibrio harveyi. Specifically, luciferase catalyzes the flavin-mediated oxidation by oxygen of a long-chain aldehyde to yield carboxylic acid and blue-green light. (.lambda.max or 490 nm:1). The reaction in vitro is initiated by injection of reduced flavin mononucleotide, FMNH.sub.2 into a vial containing the luciferase, oxygen, and a long-chain aldehyde, usually n-decyl aldehyde. The reaction pathway is as follows: ##STR1## The reaction in vivo is thought to involve a series of accessory enzymes which supply the luciferase with reduced flavin and the aldehyde substrate.
Since bacterial luciferase is an enzyme, it would appear readily capable of being produced in another organism by recombinant DNA technology. Additionally, the commercial usefulness of being able to produce bacterial luciferase in a controlled fashion employing recombinant DNA technology would be substantial. However, previous efforts to clone the luciferase genes of Beneckea (now referred to as Vibrio harveyi) in E. coli have been unsuccessful. Lamfrom H. et al., J. Bacteriology, 133, 354 (1978). These efforts were performed by producing randomly sheared fragments of Beneckea DNA and inserting them into plasmid DNA. The hybrid plasmids were then used to transform E. coli. However, the cloning efforts did not result in luciferase gene expression nor the expression of bioluminescence in E. coli.
The present invention represents a significant and distinct contribution to the art in that it provides a means for cloning luciferase genes from microorganisms such as Vibrio harveyi to produce constitutive expression of the gene in a host microorganism. As evidence of the expression of the cloned luciferase genes of the present invention, luciferase synthesized within a transformed host microorganism is of the same molecular weight as Vibrio harveyi luciferase. In addition, the bioluminescence spectra produced by a transformed host microorganism of the present invention and that of Vibrio harveyi are indistinguishable.